Both maternal IFNγ exposure and acute prenatal infection with Toxoplasma gondii activate fetal hematopoietic stem cells.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but the fetal hematopoietic response to infection during pregnancy is not well-studied. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFNγ-mediated maternal immunity. While it is known that maternal infection without direct pathogen transmission can affect fetal immune development, the effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. Furthermore, maternal IFNγ crosses the fetal-maternal interface, where it is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternal T. gondii infection, we reveal that the effects of IFNγ treatment mimic some aspects of the fetal HSC response to infection. Moreover, our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ-induced inflammation. Altogether, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors.

Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA-sequencing approach in which tissue is reversibly fixed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas profiling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

Genetic mapping reveals Nfkbid as a central regulator of humoral immunity to Toxoplasma gondii.

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.

Eukaryotic elongation factor 2 kinase regulates foam cell formation via translation of CD36.

Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase that controls protein synthesis in cells under stress. Although well studied in cancer, less is known about its roles in chronic inflammatory diseases. Here, we examined its regulation of macrophage cholesterol handling in the context of atherosclerosis. eEF2K mRNA expression and protein activity were upregulated in murine bone marrow-derived macrophages (BMDMs) exposed to oxidized low-density lipoprotein cholesterol (oxLDL). When incubated with oxLDL, BMDMs from eEF2K knockout (Eef2k-/- ) mice formed fewer Oil Red O+ foam cells than Eef2k+/+ BMDMs (12.5% ± 2.3% vs. 32.3% ± 2.0%, p < .01). Treatment with a selective eEF2K inhibitor, JAN-384, also decreased foam cell formation for C57BL/6J BMDMs and human monocyte-derived macrophages. Disabling eEF2K selectively decreased protein expression of the CD36 cholesterol uptake receptor, mediated by a reduction in the proportion of translationally active Cd36 mRNA. Eef2k-/- mice bred onto the Ldlr-/- background developed aortic sinus atherosclerotic plaques that were 30% smaller than Eef2k+/+ -Ldlr-/- mice after 16 weeks of high cholesterol diet (p < .05). Although accompanied by a reduction in plaque CD36+ staining (p < .05) and lower CD36 expression in circulating monocytes (p < .01), this was not associated with reduced lipid content in plaques as measured by oil red O staining. Finally, EEF2K and CD36 mRNA levels were higher in blood mononuclear cells from patients with coronary artery disease and recent myocardial infarction compared to healthy controls without coronary artery disease. These results reveal a new role for eEF2K in translationally regulating CD36 expression and foam cell formation in macrophages. Further studies are required to explore therapeutic targeting of eEF2K in atherosclerosis.

capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.

Translation of eukaryotic mRNAs begins with binding of their m7G cap to eIF4E, followed by recruitment of other translation initiation factor proteins. We describe capCLIP, a novel method to comprehensively capture and quantify the eIF4E (eukaryotic initiation factor 4E) 'cap-ome' and apply it to examine the biological consequences of eIF4E-cap binding in distinct cellular contexts. First, we use capCLIP to identify the eIF4E cap-omes in human cells with/without the mTORC1 (mechanistic target of rapamycin, complex 1) inhibitor rapamycin, there being an emerging consensus that rapamycin inhibits translation of TOP (terminal oligopyrimidine) mRNAs by displacing eIF4E from their caps. capCLIP reveals that the representation of TOP mRNAs in the cap-ome is indeed systematically reduced by rapamycin, thus validating our new methodology. capCLIP also refines the requirements for a functional TOP sequence. Second, we apply capCLIP to probe the consequences of phosphorylation of eIF4E. We show eIF4E phosphorylation reduces overall eIF4E-mRNA association and, strikingly, causes preferential dissociation of mRNAs with short 5'-UTRs. capCLIP is a valuable new tool to probe the function of eIF4E and of other cap-binding proteins such as eIF4E2/eIF4E3.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Fast pesticide analysis using low-pressure gas chromatography paired with a triple quadrupole mass spectrometer equipped with short collision cell technology.

RATIONALE: A proof of concept showing GC-MS/MS analysis time for pesticides can be dramatically reduced while maintaining a similar separation efficiency by combining a low-pressure gas chromatography (LPGC) column with the enhanced selected reaction monitoring (SRM) switching speed of the short collision cell of a JEOL JMS-TQ4000GC. METHODS: Triple-quadrupole tandem mass spectrometry (standard EI + at 70 eV) was used to measure pesticides eluting from a low-pressure gas chromatograph capillary column. Three transitions for each of 244 pesticide compounds were measured within an 11-min analysis time, and the data were checked to confirm the method's reproducibility and ability to distinguish all three transitions for each pesticide. RESULTS: All three transitions for all 244 pesticides were detected in the standard mixture at 1X concentration within the 11-min analysis time. Relative standard deviation (RSD) of peak areas was less than 15% for 242 pesticides, and I/Q RSDs were less than 10% for 242 compounds. Retention time RSD over 15 replicates was less than 0.1%. CONCLUSIONS: Results show that analysis time can be markedly decreased using an LPGC column, and that the ability of the short collision cell to distinguish a large number of coeluting peaks makes the two technologies a natural pairing. The effective measurement of pesticides within a short time could benefit any scientists doing pesticide analysis work.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

Bicuculline regulated protein synthesis is dependent on Homer1 and promotes its interaction with eEF2K through mTORC1-dependent phosphorylation.

The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.

Tuning Specific Translation in Cancer Metastasis and Synaptic Memory: Control at the MNK-eIF4E Axis.

The eukaryotic translation initiation factor (eIF) 4E, which binds to the 5'-cap of mRNA, undergoes phosphorylation on a single conserved serine, executed by the mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs). However, the functional consequences and physiological roles of MNK signalling have remained obscure. Now, new pharmacological and genetic tools have provided unprecedented insights into the function of MNKs and eIF4E phosphorylation. The studies suggest that MNKs control the translation of specific mRNAs in cancer metastasis and neuronal synaptic plasticity by a novel mechanism involving the regulation of the translational repressor, cytoplasmic fragile-X protein-interacting protein 1 (CYFIP1). These recent breakthroughs go a long way to resolving the longstanding enigma and controversy surrounding the function of the MNK-eIF4E axis in cancer cell biology and neurobiology.

PD-L1, TIM-3, and CTLA-4 Blockade Fails To Promote Resistance to Secondary Infection with Virulent Strains of Toxoplasma gondii.

T cell exhaustion is a state of hyporesponsiveness that develops during many chronic infections and cancer. Neutralization of inhibitory receptors, or "checkpoint blockade," can reverse T cell exhaustion and lead to beneficial prognoses in experimental and clinical settings. Whether checkpoint blockade can resolve lethal acute infections is less understood but may be beneficial in vaccination protocols that fail to elicit sterilizing immunity. Since a fully protective vaccine for any human parasite has yet to be developed, we explored the efficacy of checkpoint inhibitors in a mouse model of Toxoplasma gondii reinfection. Mice chronically infected with an avirulent type III strain survive reinfection with the type I RH strain but not the MAS, GUY-DOS, and GT1 parasite strains. We report here that mouse susceptibility to secondary infection correlates with the initial parasite burden and that protection against the RH strain is dependent on CD8 but not CD4 T cells in this model. When given a lethal secondary infection, CD8 and CD4 T cells upregulate several coinhibitory receptors, including PD-1, TIM-3, 4-1bb, and CTLA-4. Moreover, the gamma interferon (IFN-γ) response of CD8 but not CD4 T cells is significantly reduced during secondary infection with virulent strains, suggesting that checkpoint blockade may reduce disease severity. However, single and combination therapies targeting TIM-3, CTLA-4, and/or PD-L1 failed to reverse susceptibility to secondary infection. These results suggest that additional host responses, which are refractory to checkpoint blockade, are likely required for immunity to this pathogen.

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

Transcriptional and Linkage Analyses Identify Loci that Mediate the Differential Macrophage Response to Inflammatory Stimuli and Infection.

Macrophages display flexible activation states that range between pro-inflammatory (classical activation) and anti-inflammatory (alternative activation). These macrophage polarization states contribute to a variety of organismal phenotypes such as tissue remodeling and susceptibility to infectious and inflammatory diseases. Several macrophage- or immune-related genes have been shown to modulate infectious and inflammatory disease pathogenesis. However, the potential role that differences in macrophage activation phenotypes play in modulating differences in susceptibility to infectious and inflammatory disease is just emerging. We integrated transcriptional profiling and linkage analyses to determine the genetic basis for the differential murine macrophage response to inflammatory stimuli and to infection with the obligate intracellular parasite Toxoplasma gondii. We show that specific transcriptional programs, defined by distinct genomic loci, modulate macrophage activation phenotypes. In addition, we show that the difference between AJ and C57BL/6J macrophages in controlling Toxoplasma growth after stimulation with interferon gamma and tumor necrosis factor alpha mapped to chromosome 3, proximal to the Guanylate binding protein (Gbp) locus that is known to modulate the murine macrophage response to Toxoplasma. Using an shRNA-knockdown strategy, we show that the transcript levels of an RNA helicase, Ddx1, regulates strain differences in the amount of nitric oxide produced by macrophage after stimulation with interferon gamma and tumor necrosis factor. Our results provide a template for discovering candidate genes that modulate macrophage-mediated complex traits.

Instrumentation and Method Development for On-Site Analysis of Helium Isotopes.

Helium isotope determination may be useful in measuring volcanic activity and issuing earlier warnings of possible eruptions. A method is presented for measuring the 3He/4He ratio in a gas sample using a multiturn time-of-flight mass spectrometer "infiTOF". In contrast to conventional waveform averaging, peaks are determined by counting ion pulses from each time-of-flight trigger. Samples were also measured by conventional magnetic-sector mass spectrometry for comparison. Magnetic sector results were used to designate a standard for infiTOF measurement and to calculate a ratio for each sample measured by infiTOF. Mass assignment error for ultrapure 3He+ standard was 4.30 × 10-5 Da. Mass assignment error of 4He2+ and 3He+ for sample cylinders was 3.00 × 10-8 Da and 2.25 × 10-4 Da, respectively. Abundance ratios determined by infiTOF were found to be within 2% of the abundance ratios determined by magnetic-sector mass spectrometry. Mass drift was <50 × 10-6 Da over 10 h. Sample flow rate was not found to affect the results as long as the reference sample was analyzed under the same conditions. Results indicate that the infiTOF system may be a viable tool for measuring helium isotopes, which may eventually lead to earlier warnings of volcanic activity.

The genetic basis for individual differences in mRNA splicing and APOBEC1 editing activity in murine macrophages.

Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity.

Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma.

gammadelta T cells uniquely contribute to host immune defense, but how this is accomplished remains unclear. Here, we analyzed the nonclassical major histocompatibility complex class I T10 and T22-specific gammadelta T cells in mice and found that encountering antigen in the thymus was neither required nor inhibitory for their development. But when triggered through the T cell receptor, ligand-naive lymphoid-gammadelta T cells produced IL-17, whereas ligand-experienced cells made IFN-gamma. Immediately after immunization, a large fraction of IL-17(+) gammadelta T cells were found in the draining lymph nodes days before the appearance of antigen-specific IL-17(+) *beta T cells. Thus, thymic selection determines the effector fate of gammadelta T cells rather than constrains their antigen specificities. The swift IL-17 response mounted by antigen-naive gammadelta T cells suggests a critical role for these cells at the onset of an acute inflammatory response to novel antigens.

A new approach for accurate mass assignment on a multi-turn time-of-flight mass spectrometer.

A simple, effective accurate mass assignment procedure for a time-of-flight mass spectrometer is desirable. External mass calibration using a mass calibration standard together with an internal mass reference (lock mass) is a common technique for mass assignment, however, using polynomial fitting can result in mass-dependent errors. By using the multi-turn time-of-flight mass spectrometer infiTOF-UHV, we were able to obtain multiple time-of-flight data from an ion monitored under several different numbers of laps that was then used to calculate a mass calibration equation. We have developed a data acquisition system that simultaneously monitors spectra at several different lap conditions with on-the-fly centroid determination and scan law estimation, which is a function of acceleration voltage, flight path, and instrumental time delay. Less than 0.9 mDa mass errors were observed for assigned mass to charge ratios ( m/z) ranging between 4 and 134 using only 40Ar+ as a reference. It was also observed that estimating the scan law on-the-fly provides excellent mass drift compensation.

Analytical applications of electron monochromator-mass spectrometry.

An electron monochromator (EM) produces an electron beam with a narrow energy distribution that can be utilized with mass spectrometry (MS). The history and development of the EM from an initial research design to a commercial model are reviewed along with MS research applications. An EM incorporated with a mass spectrometer showed significant improvement in sensitivity over traditional methods for negative-ion generation and selectivity for compounds with electrophilic character. Sensitivity of EM-MS has been shown to be 25 fg for hexachlorobenzene in positive-ion mode and 10 fg for nitrobenzene in negative-ion mode. Reports regarding the analysis of chlorinated compounds, explosives, pesticides, phthalates, polychlorodibenzo-p-dioxins, polycyclic aromatic hydrocarbons (PAHs), nitro-polycyclic aromatic hydrocarbons (NPAHs), antioxidants, and bacterial biomarkers are discussed. Additionally, theoretical methods to predict electron-capture properties are presented.

Identification of bacteria by fatty acid profiling with direct analysis in real time mass spectrometry.

RATIONALE: Bacterial fatty acid profiling is a well-established technique for bacterial identification. Current methods involving esterification and gas chromatography/mass spectrometry (GC/MS) or matrix-assisted laser desorption/ionization (MALDI) analysis are effective, but there are potential benefits to be gained by investigating ambient ionization methods that can provide rapid analysis without derivatization or additional sample handling. METHODS: Lipid extracts from colonies of five Gram-positive and five Gram-negative pathogenic bacteria were analyzed by Direct Analysis in Real Time (DART) ionization coupled with a time-of-flight mass spectrometer. Fatty acid profiles were obtained from the negative-ion DART mass spectra without additional derivatization or sample preparation. RESULTS: Fatty acid profiles obtained from the deprotonated molecules [M - H](-) were found to be highly species-specific and reproducible. Leave-one-out cross validation (LOOCV) for principal component analysis (PCA) showed 100% correct classification accuracy. CONCLUSIONS: The results of this preliminary feasibility study show good precision and accuracy, and the fatty acid patterns are clearly distinctive for each of the ten species examined. The speed and ease of analysis and the high classification accuracy for this initial study indicate that DART is an effective method for bacterial fatty acid profiling.

Sex-Specific Expression of Histone Lysine Demethylases (KDMs) in Thyroid Cancer.

BACKGROUND: The incidence of thyroid cancer in women is 3-4-fold higher than in men. To characterize sex-specific molecular alterations in thyroid cancer, we examined the expression of sex-biased genes in normal thyroids and thyroid tumors. METHODS: Ingenuity pathways analysis was used to define sex-biased gene networks using data from the Cancer Genome Atlas (TCGA). Confirmatory studies were performed through the analysis of histone lysine demethylases (KDMs) expression by real-time PCR and immunostaining. RESULTS: In normal thyroids, 44 sex-biased genes were comparatively upregulated in male and 28 in female patients. The expressions of 37/72 (51%) sex-biased genes were affected in cancer tissues compared with normal thyroids. Gene network analyses revealed sex-specific patterns in the expressions of KDM5C, KDM5D, and KDM6A. In confirmatory studies, KDM5D mRNA and protein were detected only in males, whereas KDM5C and KDM6A were detected in samples from male and female patients. Nuclear staining with anti-KDMs was found in normal thyroids, but a loss of nuclear expression with a concomitant gain of cytoplasmic staining was observed in cancer tissues. CONCLUSIONS: Normal thyroids have a sex-specific molecular signature, and the development of thyroid cancer is associated with a differential expression of sex-biased genes. The sex-specific expression of KDMs, coupled with cancer-related alterations in their intracellular localization, may contribute to mechanisms underlying sex differences in thyroid tumorigenesis.